The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in Patients with Chronic Periodontitis

Statement of the Problem Trichomonas tenax and Entamoeba gingivalis are commensal protozoa which inhabit the human oral cavity. These parasites are found in patients with poor oral hygiene and might be a reason for progressive periodontal diseases. Purpose The aim of this study was to evaluate the effect of nonsurgical periodontal treatment on the frequency of these protozoa in saliva and plaque samples. Materials and Method In this clinical trial, samples of saliva and dental plaque were collected from 46 patients with moderate to severe chronic periodontitis before and after periodontal therapy. The samples were assessed for the frequency of parasites. Results The frequency of Entamoeba gingivalis was reduced in saliva (p= 0.007) and plaque (p= 0.027) three weeks after the treatment. Likewise, the frequency of Trichomonas tenax reduced in saliva (p= 0.030); however, the decrease was not significant in plaque (p= 0.913). Trichomonas tenax frequency in dental plaque directly related to the severity of periodontitis (r= 0.565, p≤ 0.000). In contrast, the number of Entamoeba gingivalis in both saliva (r= -0.405, p≤ 0.005) and plaque (r= -0.304, p= 0.040) was inversely related with the severity of the periodontal disease. Conclusion Nonsurgical periodontal treatment could reduce the number of Trichomonas Tenax and Entamoeba gingivalis in the oral environment of patients with chronic periodontitis.


Introduction
Presenting in several various clinical forms, periodontitis is considered as one of the most widespread oral diseases. Approximately 5 to 20 percent of the world's population suffers from severe generalized periodontitis.
[1] Periodontal lesions contain numerous neutrophils, motile bacteria, spirillae, spinning rods, and protozoa. [2] Yet, the etiology of this multi-factorial disease is still unclear. The possible role of parasites in the development of periodontitis has been poorly studied. [3] Data from previous studies about Entamoeba gingivalis and Trichomonas is also limited [4] and have been conducted only in few countries. [5] Trichomonas are mostly parasitic or commensal flagellates that live in low-oxygen environments. [6][7][8] T. tenax is currently considered as a member of the oral biofilm. [9,10] Its prevalence in the oral cavity ranges from 4 to 53% worldwide; [11] however, in patients with periodontitis; it is 3 to 4 times more than healthy individuals. [12] Investigations have been performed on the correlation between the prevalence of T. tenax and the status of periodontitis, [6,13] which is found partic-ularly in patients with poor oral hygiene. [13][14][15] It can be simply transmitted through saliva, droplet spray, kissing, using contaminated dishes, and drinking water. [4,7,11] Oral cavity is the common residence of Trichomonas species; although, it is occasionally found in the respiratory tract. [16][17][18] The significance of its presence in the respiratory tract of humans is still unclear. Pleuropulmonary trichomoniasis has been observed in bare cases. [19][20][21] A study in Egypt (2004) showed that the prevalence of pulmonary trichomoniasis was 8% (20 cases) in a total of 250 individuals. [22] Entamoeba gingivalis (E. gingivalis) is found in the oropharynx, but rarely in the head and neck lesions. [23][24][25] This microorganism is found more common in patients with poor dentition, [24] periodontal disease, and immune suppression. [26][27][28] It was the first commensal found in the human oral cavity. [12,29] E. gingivalis lives on the surface of the teeth and gum tissues.
[30] Inflammatory process produces a propitious anaerobic environment for their growth. [31] The trophozoite is measured about 10-30 µm, actively motile with multiple pseudopodia, the cytoplasm contains food vacuoles with ingested bacteria, leukocytes and epithelial cells. [29] According to some studies, this amoeba is considered as an important cause of periodontal disease. [32] E. gingivalis is an opportunistic pathogen whose association with synergistic symbiotic bacteria may cause periodontal diseases in hosts with low immunity. [33][34] Conversely, some studies have reported the general presence of E. gingivalis in disease-free individuals which was related to the amount of calculus present on the teeth. [35] As previously mentioned in several studies, these parasites exist in the oral cavity and probably play a role in development of periodontal disease. Yet, no study has investigated the patients with moderate to severe chronic periodontitis. Hence, the current study was designed to evaluate the effect of nonsurgical periodontal treatment on the level of Trichomonas tenax and Entamoeba gingivalis in dental plaques and saliva.

Materials and Method
In this randomized clinical trial (#IRCT2014071013167   The changes in parasites abundance before and after the treatment were determined by using Mann-Whitney test.

Results
Out of all the 46 patients, 33 were females and 13 were males. There were 29 patients with severe periodontitis Post-hoc test revealed no correlation between the patient's age and the frequency of T. tenax in dental plaque (p= 0.066) or saliva (p= 0.34). T. tenax in dental plaque was detected only in females (p≤ 0.005).
The frequency of E. gingivalis in dental plaque (p= 0.212) and saliva (p= 0.441) had no significant difference between various groups with different levels of education. It was found more frequently in males' saliva rather than females' (p≤ 0.005). No relationship was noticed between the age and colonization of E. gingivalis in saliva (p= 0.13) and dental plaque (p= 0.314).
Three weeks after the treatment, E. gingivalis decreased in the in case group's saliva (p= 0.007) and plaque samples (p= 0.027). T. tenax also reduced in saliva (p= 0.030); nevertheless, it did not have a significant reduction in dental plaque (p= 0.913).

Discussion
The initial treatment to eliminate pathogenic bacteria in periodontal disease is the mechanical removal of dental plaque and calculus. [37][38][39] consequently, it was supposed that these methods could help reduce parasites as much as it does for perio-pathogen bacteria. The obtained results confirmed the probable efficacy of periodontal treatment in reducing the colonization of oral parasites in dental plaque except for T.tenax. No similar study was found about the effect of periodontal treatment on the management of oral parasites; however, some studies have evaluated the prevalence of these protozoans.
Ghabanchi et al., [7] observed that E. gingivalis was more abundant than T. tenax in patients with either healthy periodontium or periodontitis, which was consistent with the results of the present study. whereas, this direct relationship was not observed in salivary T. tenax or generally in E. gingivalis. It should be noted that the level of CAL was not considered as a determining factor in the severity of periodontal disease in Ibrahim's study, [26] only the pocket depth was measured. Hence, the relationship between the parasites and the disease severity cannot be confirmed with certainty. They also reported a higher abundance of E. gingivalis in hypertensive patients and T. tenax in patients with increased use of antibiotics over the preceding 6 months. Unlike the present study, they did not exclude patients with systemic complications and found that both parasites increased in diabetic patients.
Ibrahim and Abbas observed that E. gingivalis was more frequent in 61-70 year-old and T. tenax in 21- 30 year-old patients, [26] while; the age parameter had no significance in our study. Ghabanchi et al. detected that E. gingivalis could be found in dental plaque and saliva of healthy subjects. [7] Our study showed a reverse significant correlation between the severity of periodontitis and E. gingivalis frequency, which may confirm the abundance of this parasite even in healthy conditions. In contrast, Trim et al. [35] reported that E.
gingivalis was only present in pockets deeper than 4-7 mm and this result was confirmed by Albuquerque et al. [12] and contrasted by the current study. This may be due to the differences in laboratory procedures. Hamad et al. [40] showed a positive relationship between the presence of parasite in the mouth and illiteracy or low education level which was partially confirmed by the present study. In our study, T. tenax was mostly found in illiterate patients' saliva or dental plaque, but there was no relationship between the amount of E. gingivalis and various education levels. In a study, Ullah et al. [41] reported a positive association between the prevalence of mouth parasites and poverty. The higher incidence was found among the lower class which was to some extent in line with our study. The above-mentioned study also reported that the overall prevalence of these two parasites was higher in men than women. Our study found T. tenax only in females' dental plaque. This might be attributed to the higher prevalence of T. vaginalis in females and the microscopic similarities of this species and T. tenax. [6] E. gingivalis was more prevalent in males' saliva than females'. Ullah et al. [41] included smokers, snuff dippers and participants with improper brushing habits or poor oral hygiene in their study and as expected, the presence of these confounding factors was more frequent in males rather than females.
Gharavi et al. [5] found no relationship between T. tenax infection and sex or age, but E. gingivalis was related to age higher than 20 and male sex. Contrarily, Albuquerque et al. reported no correlation between age and the presence of both parasites, which was in agreement with the results of the present study.

Conclusion
Regardless of patient's demographic characteristics, it seems that oral hygiene instructions in combination with scaling and root planning can help controlling excessive colonization of parasites, particularly E. gingivalis and T. tenax and their probable opportunistic infestation.